T. cruzi strains that are resistant to the inhibitor Mu-Phe-hPhe-vinylsulfone (K002)have been identified by Dr. Juan Engel, Department of Pathology/VAMC. In an attempt to elucidate the mechanism of resistance, susceptible and resistant cells were homogenized and the extract was assayed for the ability to hydrolyze the substrate Z-F-R-AMC in the presence and absence of K002. Our results demonstrated that while the cellular extract from the susceptible strain had an IC50 of 0.05 mM the cellular extract from the resistant strain had an IC50 of 7 mM. This suggested that the enzyme cruzain itself could be involved in the mechanism of resistance. The address this possibility, genomic DNA was extracted from both susceptible and resistant strains and the cruzain gene was amplified by the PCR using primers that flanked the published 5' and 3' e nds of the coding region. The PCR amplification product was cloned into pBluescript and two clones from susceptible organism (control) and three from resistant organisms were fully sequenced. The two clones obtained from the amplification of the susceptible strain were identical among themselves but differed from the cruzain published sequence at the following positions: L-114S, T-88A, E-73G, K-6N (in the pro-domain), S64G, I172V, E184D (in the catalytic domain) and L284F, H332Y (at the C- terminus domain). The three clones from the resistant strain differed among themselves. In addition to having the above mentioned changes, the following mutations were found: Clone RC3: S24L, L48P, D84G in the catalytic domain and F284L at the C- terminus; Clone RC4: D84G in the catalytic domain and I247S, S297F at the C-terminus; Clone RC6: A-100V in the pro-domain, D84G, R188H in the catalytic domain and I247S at the C-terminus. Computer modelling is used to show the location of the mutations on the enzyme and to predict the consequences of these mutations to the binding of the enzyme to inhibitors currently being tested.